ANALYTICAL BIOCHEMISTRY 70, 6 2 4 - 6 2 7
(1976)
On The Use of Liquid Scintillation Vials in ATP-Photometry The photometric measurement of adenosine triphosphate (ATP) using the Luciferin- Luciferase reaction is widely used in the biological sciences (1-3). This assay was developed by Strehler and McElroy (4) and was modified by Holm-Hansen (5) for use in plankton biomass studies. Instruments for the measurement of the light emitted by the reaction are commercially available, and liquid scintillation vials are commonly used as a reaction vessel (5,6). In our laboratory, the JRB ATP-Photometer is used routinely for the estimation of microbial biomass. We noticed that light, particularly from fluorescent lamps, causes phosphorescent emission by the liquid scintillation vials. The intensity of phosphorescent emission is dependent on the type of vial and the illumination to which it has been subjected. At ATP concentrations close to the limit of detection this phenomenon can introduce significant error. In an attempt to measure the phosphorescent effect, we have tested six commercially available brands of vial under varying light conditions. In addition, the quenching effects of the vials were compared. Methods
The JRB ATP-Photometer was used with a setting of 8.5 on the High-Voltage adjustment knob. Dark current was nulled to zero cpm. Luciferin-Luciferase extract and crystalline ATP were obtained from Sigma Chemical Company. Dark experiments were conducted in a photographic darkroom using Kodak amber safety lights. Illumination was measured with a Gossen foot-candle meter with scales of 0-6, 0-60, and 0-600 fc. Both plastic and glass vials were examined. Plastic vials were either polyethylene (P-1, P-2) or nylon (P-3). Glass vials were either of the low potassium type (G-I, G-2) or standard borosilicate (G-3). A study of time to maximum emission rates (saturation) upon illumination under normal laboratory conditions was performed. A comparison of phosphorescent emission rates for the different vials was carried out. Decay curves of phosphorescent emission for each vial brand were recorded. Measurement of the light transmission of the vials in the application of the ATP assay were made for each brand. Results
Under normal daylight laboratory conditions with fluorescent illumination (75 fc), emission rates for all vials reach a plateau within 15 min of light exposure. 624 Copyright © 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
625
SHORT COMMUNICATIONS
Differences in phosphorescent emission among the six brands of vial were highly significant under all light conditions studied. Figure 1 illustrates the emission rates recorded after exposure of the vials to the different light regimes for 8 hr or more. For vials P-1, P-2, G-1, and G-2, maximum counts per minute were under 200 after exposure to the highest light intensity tested (600 fc). Under normal laboratory conditions with no artificial illumination (13 fc), emission rates for these vials were consistently lower than the typical e n z y m e blank. Emission rates of the G-3 and P-3 vials were approximately an order of magnitude greater than those of the other vials tested. Figure 2 illustrates the decay of phosphorescence over a period of 1 min for the different vials tested. All vials showed a rapid initial decay of phosphorescence. H o w e v e r , vials G-3 and P-3 exhibited high residual phosphorescence at the end of the l-rain integration period. The JRB A T P - P h o t o m e t e r allows a 15-sec delay before initiation of the 1-min integration period. It is important to note that after 15 sec in the dark, light emission is substantially reduced in vials G-l, G-2, P-l, and P-2. The efficiency of light transmission varied widely among brands. As indicated in Fig. 3, counts for a standard A T P solution ranged from a maximum of 6901 cpm using the P-1 vials, to a minimum of 4246 cpm using
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FIG. 1. Rates of phosphorescence after long-term exposure to different light levels. Vials were exposed to the followingconditions for at least 12hr and then counted individuallyfor 1 min: (1) Dark, (2) 15 fc (daylight laboratory conditions), (3) 100 fc (daylight laboratory conditions with artificial illumination "cool-white" fluorescent lamps), (4) 600 fc with "cool-white" fluorescent lamps.
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FI6. 2. Decay of phosphorescence after long-term exposure. Vials were exposed to an illumination of 600 fc using "cool-white" fluorescent lamps for 48 hr. Continuous graphs of phosphorescent decay were then plotted for each vial using a millivolt recorder attached to the photometer.
the G-3 vial. In general, plastic vials transmitted light generated by the Luciferase reaction more efficiently than glass vials. Discussion
We have shown that phosphorescent emission by liquid-scintillation vials can introduce significant errors in photometric ATP analysis. Choice of vial brand and material is thus of primary concern to the investigator. Once the choice has been made, it is advisable to use only one type of vial in any series of assays. In general, light transmission by plastic vials is greater than that by glass. However, some plastic vials may exhibit unacceptably high rates of phosphorescent emission. Also of critical importance is the light regime under which the assay is performed. Our experience indicates that fluorescent laboratory illumination should be avoided and that vials should be kept in the dark before use. ACKNOWLEDGMENTS This work was supported in part by National Science Foundation Grant No. GB-31102 to Dr. K. E. Cooksey. This paper is a contribution from the Rosenstiel School of Marine and Atmospheric Science, Division of Biology and Living Resources, University of Miami, Miami, Florida 33149.
SHORT COMMUNICATIONS
627
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FIG. 3. Efficiency of light transmission +-_ 1 SD. Vials were placed in the dark for 24-48 hr before use. Standard ATP analyses were then carried out using equal volumes of ATP solution ( 10-5 mg/ml) and firefly extract ( 10 mg/ml) in a total volume of 0,4 ml. Four replicates of each vial type were used. The experiments were performed in a photographic darkroom.
REFERENCES 1. Coombs, J., Halicki, D., Holm-Hansen, O., and Volcani, B. (1967) Exp. Cell Res. 47, 302- 314. 2, Caumas, R., and Frala, M. (1969) Mar. Biol. (Berl.) 3, 243-246. 3. Cole, H. A., Wimpenny, J. W. T., and Hughes, D. F. (1967)Biochem. Biophys. Acta 143, 445-453, 4. Strehler, B. L., and McElroy, W. D. (1957) in Methods in Enzymology (Colowick S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 871-873. 5. Holm-Hansen, O., and Booth, C. (1968) Limnol. Oceanogr. 11, 510-519. 6. Hammerstedt, R. H. (1973) Anal. Biochem. 52, 449-455. JORGE E . CORREDOR DOUGLAS G. CAPONE K E I T H E . COOKSEY
Division of Biology and Living Resources Rosenstiel School of Marine and Atmospheric Science University of Miami 4600 Rickenbacker Causeway Miami, Florida 33149 Received April 17, 1975; accepted September 2, 1975
(1976)
On The Use of Liquid Scintillation Vials in ATP-Photometry The photometric measurement of adenosine triphosphate (ATP) using the Luciferin- Luciferase reaction is widely used in the biological sciences (1-3). This assay was developed by Strehler and McElroy (4) and was modified by Holm-Hansen (5) for use in plankton biomass studies. Instruments for the measurement of the light emitted by the reaction are commercially available, and liquid scintillation vials are commonly used as a reaction vessel (5,6). In our laboratory, the JRB ATP-Photometer is used routinely for the estimation of microbial biomass. We noticed that light, particularly from fluorescent lamps, causes phosphorescent emission by the liquid scintillation vials. The intensity of phosphorescent emission is dependent on the type of vial and the illumination to which it has been subjected. At ATP concentrations close to the limit of detection this phenomenon can introduce significant error. In an attempt to measure the phosphorescent effect, we have tested six commercially available brands of vial under varying light conditions. In addition, the quenching effects of the vials were compared. Methods
The JRB ATP-Photometer was used with a setting of 8.5 on the High-Voltage adjustment knob. Dark current was nulled to zero cpm. Luciferin-Luciferase extract and crystalline ATP were obtained from Sigma Chemical Company. Dark experiments were conducted in a photographic darkroom using Kodak amber safety lights. Illumination was measured with a Gossen foot-candle meter with scales of 0-6, 0-60, and 0-600 fc. Both plastic and glass vials were examined. Plastic vials were either polyethylene (P-1, P-2) or nylon (P-3). Glass vials were either of the low potassium type (G-I, G-2) or standard borosilicate (G-3). A study of time to maximum emission rates (saturation) upon illumination under normal laboratory conditions was performed. A comparison of phosphorescent emission rates for the different vials was carried out. Decay curves of phosphorescent emission for each vial brand were recorded. Measurement of the light transmission of the vials in the application of the ATP assay were made for each brand. Results
Under normal daylight laboratory conditions with fluorescent illumination (75 fc), emission rates for all vials reach a plateau within 15 min of light exposure. 624 Copyright © 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
625
SHORT COMMUNICATIONS
Differences in phosphorescent emission among the six brands of vial were highly significant under all light conditions studied. Figure 1 illustrates the emission rates recorded after exposure of the vials to the different light regimes for 8 hr or more. For vials P-1, P-2, G-1, and G-2, maximum counts per minute were under 200 after exposure to the highest light intensity tested (600 fc). Under normal laboratory conditions with no artificial illumination (13 fc), emission rates for these vials were consistently lower than the typical e n z y m e blank. Emission rates of the G-3 and P-3 vials were approximately an order of magnitude greater than those of the other vials tested. Figure 2 illustrates the decay of phosphorescence over a period of 1 min for the different vials tested. All vials showed a rapid initial decay of phosphorescence. H o w e v e r , vials G-3 and P-3 exhibited high residual phosphorescence at the end of the l-rain integration period. The JRB A T P - P h o t o m e t e r allows a 15-sec delay before initiation of the 1-min integration period. It is important to note that after 15 sec in the dark, light emission is substantially reduced in vials G-l, G-2, P-l, and P-2. The efficiency of light transmission varied widely among brands. As indicated in Fig. 3, counts for a standard A T P solution ranged from a maximum of 6901 cpm using the P-1 vials, to a minimum of 4246 cpm using
i0oo,
Eoo.lO0
'
o 3
,
oo
,
i
J
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~oo 300 400 500 ILLUMINATION ( foot -candles
i
600
FIG. 1. Rates of phosphorescence after long-term exposure to different light levels. Vials were exposed to the followingconditions for at least 12hr and then counted individuallyfor 1 min: (1) Dark, (2) 15 fc (daylight laboratory conditions), (3) 100 fc (daylight laboratory conditions with artificial illumination "cool-white" fluorescent lamps), (4) 600 fc with "cool-white" fluorescent lamps.
626
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FI6. 2. Decay of phosphorescence after long-term exposure. Vials were exposed to an illumination of 600 fc using "cool-white" fluorescent lamps for 48 hr. Continuous graphs of phosphorescent decay were then plotted for each vial using a millivolt recorder attached to the photometer.
the G-3 vial. In general, plastic vials transmitted light generated by the Luciferase reaction more efficiently than glass vials. Discussion
We have shown that phosphorescent emission by liquid-scintillation vials can introduce significant errors in photometric ATP analysis. Choice of vial brand and material is thus of primary concern to the investigator. Once the choice has been made, it is advisable to use only one type of vial in any series of assays. In general, light transmission by plastic vials is greater than that by glass. However, some plastic vials may exhibit unacceptably high rates of phosphorescent emission. Also of critical importance is the light regime under which the assay is performed. Our experience indicates that fluorescent laboratory illumination should be avoided and that vials should be kept in the dark before use. ACKNOWLEDGMENTS This work was supported in part by National Science Foundation Grant No. GB-31102 to Dr. K. E. Cooksey. This paper is a contribution from the Rosenstiel School of Marine and Atmospheric Science, Division of Biology and Living Resources, University of Miami, Miami, Florida 33149.
SHORT COMMUNICATIONS
627
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2000-
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FIG. 3. Efficiency of light transmission +-_ 1 SD. Vials were placed in the dark for 24-48 hr before use. Standard ATP analyses were then carried out using equal volumes of ATP solution ( 10-5 mg/ml) and firefly extract ( 10 mg/ml) in a total volume of 0,4 ml. Four replicates of each vial type were used. The experiments were performed in a photographic darkroom.
REFERENCES 1. Coombs, J., Halicki, D., Holm-Hansen, O., and Volcani, B. (1967) Exp. Cell Res. 47, 302- 314. 2, Caumas, R., and Frala, M. (1969) Mar. Biol. (Berl.) 3, 243-246. 3. Cole, H. A., Wimpenny, J. W. T., and Hughes, D. F. (1967)Biochem. Biophys. Acta 143, 445-453, 4. Strehler, B. L., and McElroy, W. D. (1957) in Methods in Enzymology (Colowick S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 871-873. 5. Holm-Hansen, O., and Booth, C. (1968) Limnol. Oceanogr. 11, 510-519. 6. Hammerstedt, R. H. (1973) Anal. Biochem. 52, 449-455. JORGE E . CORREDOR DOUGLAS G. CAPONE K E I T H E . COOKSEY
Division of Biology and Living Resources Rosenstiel School of Marine and Atmospheric Science University of Miami 4600 Rickenbacker Causeway Miami, Florida 33149 Received April 17, 1975; accepted September 2, 1975
